CN107356744A - A kind of method and its kit of circulating tumor cell sorting and/or enrichment - Google Patents
A kind of method and its kit of circulating tumor cell sorting and/or enrichment Download PDFInfo
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- CN107356744A CN107356744A CN201710568652.9A CN201710568652A CN107356744A CN 107356744 A CN107356744 A CN 107356744A CN 201710568652 A CN201710568652 A CN 201710568652A CN 107356744 A CN107356744 A CN 107356744A
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Abstract
The invention provides the method for a kind of sorting of circulating tumor cell and/or enrichment, method provided by the invention utilizes affinity high and stable between the anti-Her2 monoclonal antibodies of biotin labeling, the anti-Trop2 monoclonal antibodies of the anti-EpCAM monoclonal antibody of biotin labeling and biotin labeling and the coated magnetic bead of Avidin, there is provided a kind of immunomagnetic beads method sorts and/or enrichment cycles tumour cell.Present invention also offers a kind of kit for sorting and/or being enriched with for tumour cell.The method of the present invention can delicately capture tumour cell, be screened suitable for the broad spectrum activity of tumour cell, and overcome the steric effect defect of common magnetic bead screening, reach the effect of efficient capture tumour cell.
Description
Technical field
The invention belongs to biomedicine field, in particular it relates to a kind of sorting of circulating tumor cell and/or enrichment
Method, the invention further relates to it is a kind of for circulating tumor cell sort and/or be enriched with kit.
Background technology
Circulating tumor cell (circulating tumor cell, CTC) is to shed into sanguimotor tumour cell,
Many experiments have confirmed that CTC detections contribute to tumor recurrence transfer monitoring, judge patient's prognosis, instruct postoperative adjuvant therapy
Deng.But quantity of the CTC in peripheral blood is few, only have generally in about 100,000,000 leucocytes and 50,000,000,000 red blood cells several swollen
Oncocyte.Therefore, in order to improve the recall rate of circulating tumor cell, it usually needs carry out the richness of circulating tumor cell before detection
Collection.The enrichment of circulating tumor cell can be separated (immune separation) or be utilized by using the Specific marker of tumour cell
The difference (such as cell volume and density) of cell morphological characteristic is realized.
Conventional cell enrichment technology mainly has immunological magnetic bead sorting, density gradient centrifugation and cell filtration etc..Immune magnetic
Pearl sorting be by antigen-antibody combine based on circulating tumor cell separation method, be that the most frequently used method is (such as at present
CN201110005376.8, a kind of method of the separation and concentration target cell from tissue；The detection of circulating tumor cell and its in breast
Clinical practice in adenocarcinoma patients, Ma Deliang etc., Journal of Clinical Oncology, in November, 2010 o. 11th of volume 15；Circulating tumor
The detection of cell, Li Shuai etc., Medical review, the 6th phase of volume 16 in March, 2010) etc., its general principle be using tumour cell and
The different surfaces antigenic mark of normal blood cell expression, designs that different antibody is in combination, utilizes what antigen-antibody combined
High degree of specificity and sensitivity, the purpose for reaching difference and enrichment cycles tumour cell (is to combine antibody and magnetic bead mostly, in magnetic
Reach the purpose of separation in).Antibody immune magnetic beads are to be combined specific monoclonal antibody with magnetic bead, and cell surface antigen can be with connecting
The specific monoclonal antibody for being connected to magnetic bead is combined, and in externally-applied magnetic field, the cell being connected by antibody with magnetic bead is adsorbed and delay
In magnetic field, the cell of no respective surfaces antigen with being connected to the specific monoclonal antibody of magnetic bead due to can not be combined without magnetic
Property, do not stopped in magnetic field, so that cell separates.
But the direct coupling generally use NHS- active ester methods of current antibody and magnetic bead, that is, the carboxylated magnetic bead activated
Reacted with the primary amino groups of antibody.Wherein, the electronegativity of amino is stronger, and coupling efficiency is higher.But Charged acids are often
Play the part of important role in antibody structure stabilization and in the cohesive process of antibody and antigen.These key amino acids and magnetic bead
Crosslinking can have a strong impact on the stability of antibody structure and the activity of antibody.In addition, in the method that magnetic bead-antibody is directly coupled,
Magnetic bead is directly crosslinked with the amino acid on antibody, because the steric hindrance of magnetic bead can influence the structure of antibody, easily causes antibody
Hydrophobic region it is exposed, easily cause the focusing of antibody and lose activity, so as to influence the stability of its product.It is additionally, since outer
Circulating tumor cell (CTC) rare numbers in all blood, and have the characteristics that heterogeneous and easily assemble agglomerating.This method is sensitive
There is also problems for property, specificity and repeatability etc..
Application No. CN201410497998.0 Chinese patent application discloses a kind of antibody immune magnetic beads method, and it is public
Combination three kinds of anti-HLA-G, EpCAM and CK8/18 monoclonal antibody coupling immunomagnetic beads answering in tumour cell sorting are opened
With.But this method uses the aldehyde radical coupling method without selectivity.Aldehyde radical can both react with amino, can also be anti-with carboxyl
Should.This method is frequently used in the coupling reaction between albumen and albumen.Poly crosslinking between albumen and albumen can pass through
The methods of ultracentrifugation/molecular sieve and ultrafiltration, removes.Because the course of reaction of this method is uncontrollable, and the crosslinking of antibody itself
Can not effectively it remove, antibody and magnetic bead coupling typically will not be in this way.The uncontrollable of its course of reaction can cause antibody
It is low with the coupling efficiency of magnetic bead, and difference between batches can be larger.Therefore, in the production application of reality, this method
It is difficult to produce stable product.In addition, the direct coupling of antibody and magnetic bead can cause magnetic bead to have stronger steric hindrance effect to antibody
Should, it is larger to the activity influence of antibody after crosslinking, easily cause tumour cell capture rate impacted.Its three kinds of antibody optimized
Combination ratio is also without repeatability.
Based on this, currently to the circulating tumor cell enrichment of the repeatability height, efficiency high that can be used for production application
Demand be present in method.
The content of the invention
Therefore, in order to solve the deficiencies in the prior art, it is an object of the invention to provide a kind of sorting of circulating tumor cell
And/or enrichment method, method provided by the invention using biotin labeling anti-Her2, EpCAM and Trop2 antibody with it is affine
High and stable affinity between the coated magnetic bead of element, there is provided a kind of immunomagnetic beads method sorts and/or enrichment cycles
Tumour cell.Method provided by the invention drastically increases the capture rate of circulating tumor cell.Another object of the present invention
It is to provide a kind of kit for sorting and/or being enriched with for tumour cell.
On the one hand, the invention provides a kind of circulating tumor cell sorting and/or enrichment method, methods described include with
Lower step：
1) biotin labeling anti-Her2 (human epidermal growth factor receptor 2) monoclonal antibody is used, (epithelium is thin for anti-EpCAM
Intercellular adhesion molecule) monoclonal antibody and anti-Trop2 (human trophocyte cell's surface antigen 2) monoclonal antibody, and be mixed；
Wherein, the mixed mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 20-40% of biotin labeling；
The anti-EpCAM monoclonal antibody 10-25% of biotin labeling；
The anti-Trop2 monoclonal antibodies 40-60% of biotin labeling；
2) mixture for obtaining step 1) is added in cell liquid to be sorted；
Wherein, the volume ratio of the mixture and cell liquid is 1~5:1000；
3) magnetic bead of Streptavidin coupling is added, is incubated 20 minutes at 4 DEG C, magnetic bead is collected, produces；
Wherein, the volume ratio of the cell liquid to be sorted and magnetic bead is 1000:10~30.
Preferably, in step 1), the mixed mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 35% of biotin labeling；
The anti-EpCAM monoclonal antibody 20% of biotin labeling；
The anti-Trop2 monoclonal antibodies 45% of biotin labeling；
Preferably, in step 2), the volume ratio of the mixture and cell liquid is 2~3:1000；
Preferably, in step 3), the volume ratio of the cell liquid to be sorted and magnetic bead is 1000:20.Preferably, originally
The described tumour of invention be selected from breast cancer, cancer of the esophagus, non-small cell lung cancer, cervix cancer, colon cancer, stomach cancer, uterine cancer and/or
Nasopharyngeal carcinoma.
Preferably, selected from EBA-1, (name of product is Ep-CAM Antibody (EBA- to the anti-EpCAM monoclonal antibody
1), production code member sc-66020), (name of product is Ep-CAM Antibody (AUA1), production code member sc- to AUA1
53277) or C-10 (name of product is Ep-CAM Antibody (C-10), production code member sc-25308), derive from
Santa Cruz Biotechnology companies), the monoclonal antibody of the anti-Her2 is selected from 24D2 (name of product Neu
Antibody (24D2), production code member sc-23864), 0.N.211 (name of product is Neu Antibody (0.N.211), production
Product numbering is sc-71667) or 3B5 (name of product is Neu Antibody (3B5), production code member sc-33684), equal source
In Santa Cruz Biotechnology companies), the monoclonal antibody of the anti-Trop2 is selected from 162-46, and (name of product is
BV421Mouse Anti-Human Trop-2Clone 162-46, product article No. for 563243), (name of product is B-9
TROP-2Antibody (B-9), production code member sc-376746) or F-5 (name of product is TROP-2Antibody (F-5),
Production code member is sc-376181), wherein 162-46 derives from Santa Cruz from BD Bioscience, B-9, F-5
Biotechnology companies).
Preferably, in step 1), the monoclonal antibodies of three kinds of biotin labelings passes through the side that comprises the following steps
It is prepared by method：
A. ultrafiltration post antibody purification solution is used；
B. NHS-PEG4-Biotin solution is added, reacts 0.5~4h at room temperature；
Wherein, the molar concentration ratio of NHS-PEG4-Biotin and antibody is 10-50:1；
The 0.5-3h preferably under room temperature reaction；1h is more preferably reacted at room temperature；
C. the monoclonal antibody solution for remove free biotin, obtaining biotin labeling is isolated and purified.
Preferably, described isolate and purify is carried out using sephadex.
Preferably, the step a is completed by the method comprised the following steps：
1. prepare mark reaction solution；
The label solution solution includes following components：
NaCl：10-300mmol/L；KCl：1-5mmol/L；Na2HPO4：5-50mmol/L；KH2PO4：1-5mmol/L；
Preferably, the mark reaction solution includes following components：
NaCl：137mmol/L；KCl：2.7mmol/L；Na2HPO4：10mmol/L；KH2PO4：2mmol/L；
2. adding mark reaction solution in ultrafiltration post, monoclonal antibody is then added, is mixed, is then centrifuged for, abandons filtrate；
Wherein, the volume mass ratio of the label solution and the monoclonal antibody is 1:1-50(μl/μg)；
Preferably, repeat step 2~3 times；
3. mixing the liquid of the residual in ultrafiltration post, after being stored at room temperature, the reversion of ultrafiltration post is inverted in a new super filter tube
In, filtrate is collected by centrifugation, takes PBS to be mixed in ultrafiltration post, stands；Ultrafiltration post is inverted, filtrate is collected by centrifugation；
In a preferred embodiment, the monoclonal antibody of the biotin labeling passes through the side that comprises the following steps
It is prepared by method：
200 μ l mark reaction solutions (NaCl 137mmol/L are added in ultrafiltration post；KCl 2.7mmol/L；Na2HPO4
10mmol/L；KH2PO42mmol/L), 500 μ g monoclonal antibodies are added, are mixed；3500g centrifuges 2min under the conditions of 4 DEG C, abandons filter
Liquid；100 μ l mark reaction solutions (NaCl137mmol/L is added in ultrafiltration post；KCl 2.7mmol/L；Na2HPO4 10mmol/
L；KH2PO42mmol/L), mix, 5000g centrifugations 2min under the conditions of 4 DEG C；The above-mentioned 100 μ l that add in ultrafiltration post are repeated to mark
Reaction solution, mix, under the conditions of 4 DEG C, 5000g centrifuges 2min step 2 time；
The liquid of the residual in ultrafiltration post is mixed, is stored at room temperature 1min；The reversion of ultrafiltration post is inverted in a new super filter tube
In, 1000g centrifuges 2min under the conditions of 4 DEG C, collects filtrate；Take 50 μ l PBS to be mixed in ultrafiltration post, stand 1min；It is inverted ultrafiltration
Post, 3500g centrifugations 2min, collects filtrate under the conditions of 4 DEG C；Above-mentioned filtrate is merged, 4 DEG C of placements are standby.With mark reaction solution
(NaCl 137mmol/L；KCl 2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L) regulation antibody concentration arrives
2mg/ml(0.25ml)。
Filtrate after ultrafiltration adds NHS-PEG4-Biotin solution (0.1-0.5mmol/L), reacts at room temperature 1h.Glucan
Gel isolates and purifies, and removes free biotin；Obtain the monoclonal antibody solution of biotin labeling.
Preferably, in step 2), the anti-Her2 monoclonal antibodies of the biotin labeling, biotin labeling it is anti-
The final concentration that EpCAM monoclonal antibodies, the anti-Trop2 monoclonal antibodies of biotin labeling use in cell liquid is respectively 300 μ
g/ml-600μg/ml。
Preferably, the anti-Her2 monoclonal antibodies of the biotin labeling, the anti-EpCAM monoclonal of biotin labeling resist
The final concentration that body, the anti-Trop2 monoclonal antibodies of biotin labeling use in cell liquid is respectively 500 μ g/ml.
Preferably, the anti-Her2 monoclonal antibodies of the biotin labeling are 24D2, the biotin of biotin labeling
The anti-EpCAM monoclonal antibody of mark is the EBA-1 of biotin labeling, the anti-Trop2 monoclonal antibodies of the biotin labeling
For the 162-46 of biotin labeling (24D2, EBA-1,162-46 derive from Santa Cruz Biotechnology companies).
On the other hand, the invention provides a kind of kit for sorting and/or being enriched with for circulating tumor cell, the examination
Agent box includes anti-Her2 monoclonal antibodies, the anti-EpCAM monoclonal antibody of biotin labeling and the biotin mark of biotin labeling
The mixture of the anti-Trop2 monoclonal antibodies of note.
Preferably, the mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 20-40% of biotin labeling；
The anti-EpCAM monoclonal antibody 10-25% of biotin labeling；
The anti-Trop2 monoclonal antibodies 40-60% of biotin labeling；
Preferably, the mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 35% of biotin labeling；
The anti-EpCAM monoclonal antibody 20% of biotin labeling；
The anti-Trop2 monoclonal antibodies 45% of biotin labeling；
Preferably, kit of the invention also includes marked by streptavidin magnetic bead (source company：BD Bioscience,
Article No.：557812).
Another further aspect, present invention also offers the method for the present invention and kit are thin in sorting and/or enrichment cycles tumour
Purposes in born of the same parents.
Compared with prior art, technical scheme has advantages below：
1) method for the three kinds of monoclonal antibodies sorting circulating tumor cell being coupled the present invention relates to a kind of combination biotin,
The present invention uses the mixture of anti-EpCAM, anti-Her2 and anti-Trop2 monoclonal antibodies, and it is thin that they can delicately capture tumour
Born of the same parents, screened suitable for the broad spectrum activity of tumour cell, and overcome the steric effect defect of common magnetic bead screening, reached and efficiently catch
Obtain the effect of tumour cell.
2) it is known to those skilled in the art that because the efficiency that various antibody are combined with tumor-cell antigen is inconsistent,
Three kinds of antibody of combination carry out tumor cell enrichment, can directly affect the capture effect of tumour cell, and three kinds of antibody combinations also understand that
This influences joint efficiency.But present inventors discovered unexpectedly that, of the invention three kinds of monoclonal antibodies are in suitable matter
Measuring in percentage range and under rational final concentration, can farthest expand the coverage rate of tumour cell, so as to greatly carry
High efficiency, and saved cost.
3) immunomagnetic beads that the present invention is formed using the monoclonal antibody of biotin labeling and the magnetic bead of Avidin is compound
Thing substitutes the method that the antibody-magnetic bead used in the prior art is directly crosslinked, and is effectively guaranteed the activity of immunomagnetic beads, improves
To the capture rate of tumour cell.The additional proportion of the method applied in the present invention antibody and magnetic bead is 4:1 to 1:Between 1, greatly
The antibody as core reagent is saved greatly.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Anti-EpCAM monoclonal antibody is EBA-1, AUA1 and C-10 in the present invention, is purchased from SANTA CRUZ
BIOTECHNOLOGY；Anti- Her2 monoclonal antibodies are 24D2,0.N.211 and 3B5, are purchased from SANTA CRUZ
BIOTECHNOLOGY；Anti- Trop2 monoclonal antibodies are 162-46, B-9 and F-5, and wherein 162-46 is purchased from BD Bioscience,
B-9 or F-5 is purchased from SANTA CRUZ BIOTECHNOLOGY.Magnetic bead in the present invention is purchased from BD Bioscience, article No.：
557812。
Embodiment 1 is combined three kinds of monoclonal antibodies sorting tumour cell of biotin coupling
1st, the preparation of the monoclonal antibody of biotin labeling
The preparation of the anti-EpCAM monoclonal antibody of 1.1 biotin labelings
In ultrafiltration post (Millipore, article No.：UFC501096 200 μ l mark reaction solutions (NaCl is added in)
137mmol/L；KCl 2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), 500 μ g anti-EpCAMs Dan Ke are added
Grand antibody (EBA-1), mix.3500g centrifuges 2min under the conditions of 4 DEG C, abandons filtrate；100 μ l mark reactions are added in ultrafiltration post
Solution (NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), mix, 4 DEG C of conditions
Lower 5000g centrifuges 2min；Repeat the above-mentioned 100 μ l that added in ultrafiltration post and mark reaction solution, mix, under the conditions of 4 DEG C, 5000g
Centrifuge 2min step 2 time；
The liquid of the residual in ultrafiltration post is mixed, is stored at room temperature 1min.The reversion of ultrafiltration post is inverted in a new super filter tube
In, 1000g centrifuges 2min under the conditions of 4 DEG C, collects filtrate.Take 50 μ l PBS to be mixed in ultrafiltration post, stand 1min.It is inverted ultrafiltration
Post, 3500g centrifugations 2min, collects filtrate under the conditions of 4 DEG C.Above-mentioned filtrate is merged, 4 DEG C of placements are standby.With mark reaction solution
(NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L) regulation antibody concentration arrives
2mg/ml(0.25ml)。
Filtrate after ultrafiltration adds NHS-PEG4-Biotin solution (0.1-0.5mmol/L), reacts at room temperature 1h.Glucan
Gel isolates and purifies (removal free biotin), obtains the monoclonal antibody solution A of biotin labeling.
The preparation of the Monoclonal Antibody Against Her2 antibody of 1.2 biotin labelings
In ultrafiltration post (Millipore, article No.：UFC501096 200 μ l mark reaction solutions (NaCl is added in)
137mmol/L；KCl 2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), the anti-Her2 monoclonals of 500 μ g are added
Antibody (24D2), mix.3500g centrifuges 2min under the conditions of 4 DEG C, abandons filtrate；100 μ l mark reaction solutions are added in ultrafiltration post
(NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), mix, under the conditions of 4 DEG C
5000g centrifuges 2min；Repeat the above-mentioned 100 μ l that added in ultrafiltration post and mark reaction solution (NaCl 137mmol/L；KCl
2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), mix, under the conditions of 4 DEG C, 5000g centrifuges 2min step 2
It is secondary；
The liquid of the residual in ultrafiltration post is mixed, is stored at room temperature 1min.The reversion of ultrafiltration post is inverted in a new super filter tube
In, 1000g centrifuges 2min under the conditions of 4 DEG C, collects filtrate.Take 50 μ l PBS to be mixed in ultrafiltration post, stand 1min.It is inverted ultrafiltration
Post, 3500g centrifugations 2min, collects filtrate under the conditions of 4 DEG C.Above-mentioned filtrate is merged, 4 DEG C of placements are standby.With mark reaction solution
(NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L) regulation antibody concentration arrives
2mg/ml(0.25ml)。
Filtrate after ultrafiltration adds NHS-PEG4-Biotin solution (0.1-0.5mmol/L), reacts at room temperature 1h.Glucan
Gel isolates and purifies (removal free biotin), obtains the monoclonal antibody solution B of biotin labeling.
The preparation of the Monoclonal Antibody Against Trop2 antibody of 1.3 biotin labelings
In ultrafiltration post (Millipore, article No.：UFC501096 200 μ l mark reaction solutions (NaCl is added in)
137mmol/L；KCl 2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), the anti-Trop2 Dan Ke of 500 μ g are added
Grand antibody (162-46), mix.3500g centrifuges 2min under the conditions of 4 DEG C, abandons filtrate；100 μ l mark reactions are added in ultrafiltration post
Solution (NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), mix, 4 DEG C of conditions
Lower 5000g centrifuges 2min；Repeat the above-mentioned 100 μ l that added in ultrafiltration post and mark reaction solution (NaCl 137mmol/L；KCl
2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L), mix, under the conditions of 4 DEG C, 5000g centrifuges 2min step 2
It is secondary；
The liquid of the residual in ultrafiltration post is mixed, is stored at room temperature 1min.The reversion of ultrafiltration post is inverted in a new super filter tube
In, 1000g centrifuges 2min under the conditions of 4 DEG C, collects filtrate.Take 50 μ l PBS to be mixed in ultrafiltration post, stand 1min.It is inverted ultrafiltration
Post, 3500g centrifugations 2min, collects filtrate under the conditions of 4 DEG C.Above-mentioned filtrate is merged, 4 DEG C of placements are standby.With mark reaction solution
(NaCl 137mmol/L；KCl2.7mmol/L；Na2HPO410mmol/L；KH2PO42mmol/L) regulation antibody concentration arrives
2mg/ml(0.25ml)。
Filtrate after ultrafiltration adds NHS-PEG4-Biotin solution (0.1-0.5mmol/L), reacts at room temperature 1h.Glucan
Gel isolates and purifies (removal free biotin), obtains the monoclonal antibody solution C of biotin labeling.
2nd, the activity of flow cytometry detection antibody
SKBR3 cells are Her2 receptor positive cells system, can be combined with Her2 antibody specificities；MCF-7 cells are
Epcam receptor positive cells system, it can be combined with Epcam antibody specificities；H1650 cells are Trop2 receptor positive cells
System, can combine with Trop2 antibody specificities；, will in order to verify the activity of Her2 antibody, Epcam antibody, Trop2 antibody
Monoclonal antibody solution A, B and the C obtained in the preparation process of the monoclonal antibody of biotin labeling respectively with SKBR3 cells,
MCF-7 cells, H1650 cell incubations, binding signal is detected using flow cytometry.
2.1 fixers are prepared：Cell-preservation liquid A (250 μ l) and cell-preservation liquid B (250 μ l) is mixed and kept away after room temperature
Light places 15min, stand-by.
2.2SKBR3 cells, MCF-7 cells, H1650 cells are collected：With 2ml 0.25% trypsin digestion cell, add
5ml culture mediums terminate, and 1000rpm centrifugation 5min, abandon supernatant, cell is resuspended in the PBS for adding 500ul.
2.3SKBR3 cells, MCF-7 cells, H1650 cells are fixed：500 μ l cell suspensions and 500ul fixers are mixed
It is even, in room temperature avoid light place 1h.700g, 5min are centrifuged, and abandon supernatant, add 1ml combination buffers and cell progress cell is resuspended
Count, and cell is diluted to 2.8 × 10 with combination buffer5Individual/ml.
2.4 antibody dilute：Antibody gradient dilution is carried out with combination buffer.
2.5 antibody incubation：The antibody diluted is mixed by 100ul/ pipes and 100 μ l cell suspensions (about 2.8 ten thousand), in
4 DEG C of concussions are incubated 30min.
2.6 cells clean：Often pipe adds 800ul PBST, 700g centrifugation 5min, and suction abandons 900 μ l supernatants, adds 900 μ l
PBST, 700g centrifuge 5min, and 900 μ l supernatants are abandoned in suction.
2.7 secondary antibodies are incubated：Often pipe adds 100ul secondary antibodies, and 30min is incubated in 4 DEG C of lucifuge concussions.
2.8 repeat steps 2.6 carry out cell cleaning.
Machine testing on 2.9：Cell is resuspended, is detected with flow cytometer.
2.9 data analysis：Data are handled with Softmax5.3 softwares.
Delta F%=(experimental group fluorescent value-feminine gender group fluorescent value)/negative group fluorescent value * 100%.Represent definitely glimmering
The percentage of the relatively negative value of light value.
3rd, tumour cell sorting and/or enrichment
Breast cancer cell (MCF-7, purchased from Chinese Academy of Sciences cell bank), lung carcinoma cell of the laboratory cultures from the Chinese Academy of Sciences
(H1650, purchased from Chinese Academy of Sciences's cell bank), colon cancer cell (SW480, purchased from Chinese Academy of Sciences's cell bank).Cell is dilute
Release to 50000/ml, per 4.8 μ l of 8ml blood addition, totally 240/pipe.
10ml fixers, fixed more than 24h are added in 8ml blood samples.Blood sample 700g centrifuge 10min, after go
Supernatant.Add 45ml erythrocyte cracked liquids, room temperature rotation cracking 15min.500g centrifuges 5min, removes supernatant.Add combination buffer
Cell is resuspended in 10ml, and once, 300g centrifuges 5min to cleaning cell.Supernatant is removed, adds appropriate cleaning combination buffer, is settled to
1ml.Be proportionally added into the mixtures of antibodies of tri- kinds of biotin labelings of A, B, C, FITC marks anti-CK antibody and APC mark it is anti-
The anti-Her2 antibody of CD45 antibody, wherein biotin labeling, the anti-EpCAM antibody of biotin labeling, biotin labeling it is anti-
The final concentration of 500 μ g/ml that Trop2 mixtures of antibodies uses in cell liquid, 4 DEG C of rotations are incubated 30min, add cleaning buffer solution
10ml cleanings cell 1 time, supernatant is removed after 300g centrifugations.Add what is be made up of the magnetic bead and FITC reinforcing agents of marked by streptavidin
The volume ratio of mixed solution 1ml, cell liquid to be sorted and magnetic bead is 1000：20,4 DEG C of rotations are incubated 20min.It is thin to carry out tumour
Born of the same parents capture.
3.1 cell dyeing interpretations of result
Observe the staining conditions of cell under the microscope by fluorescence antibody, tumour cell is counted, calculate recovery
Rate.
The analysis of Leica DM6000M microscope scanning results：Blue-fluorescence is presented to have identified with DAPI dye nucleus
Nucleus, leucocyte expression CD45, tumour cell do not express CD45, and the CD45 antibody of serviceable indicia identifies leucocyte, CK master
Epithelial cell is distributed in, therefore CK8+CD45- cell is tumour cell, CK8-CD45+ cell is leucocyte.
The selection of 2 three kinds of antibody of embodiment
Biotin antibody preparation method and the activity methods of flow cytometry detection antibody as described in Example 1, wherein raw
The anti-Her2 monoclonal antibodies of thing element mark, the anti-EpCAM monoclonal antibody of biotin labeling, the anti-Trop2 of biotin labeling
The Delta F% values of monoclonal antibody are as shown in table 1 below -3：
The Her2 antibody binding SKBR3 cell Delta F% of table 1 are counted
The Epcam antibody binding MCF-7 cell Delta F% of table 2 are counted
The Trop2 antibody binding H1650 cell Delta F% of table 3 are counted
From table 1, table 2, table 3 it is known that for Her2 monoclonal antibodies, clone number is 24D2 Her2 antibody effects
It is best.It is best for EBA-1 Epcam antibody effects for Epcam monoclonal antibodies, clone number.Resist for Trop2 monoclonals
Body, clone number are best for 162-46 Trop2 antibody effects.
The determination of the different monoclonal antibody mixed proportion of 3 three kinds of embodiment
As described in Example 1, wherein the anti-Her2 monoclonal antibodies (24D2) of biotin labeling, biotin labeling it is anti-
EpCAM monoclonal antibodies (EBA-1), biotin labeling anti-Trop2 monoclonal antibodies (162-46) mixed proportion such as table 4 below
It is shown：
Table 4:
Conclusion：Group 1, group 4,5 tumor cell recoveries of group are up to more than 70%, wherein 1 best results are organized, group 2,3 tumours of group
Cell recoveries are low, undesirable.
The determination of the mixtures of antibodies dosage of embodiment 4
Experimental program as described in Example 1, the wherein anti-Her2 monoclonal antibodies (24D2) of biotin labeling, biotin mark
The anti-EpCAM monoclonal antibody (EBA-1) of note, the mixed matter of anti-Trop2 monoclonal antibodies (162-46) of biotin labeling
It is respectively 35%, 20%, 45% to measure percentage.The final concentration that above-mentioned mixtures of antibodies uses in cell liquid is as shown in table 5 below
The determination of the mixtures of antibodies dosage of table 5
Conclusion：Group 2, group 3,4 tumor cell recoveries of group reach more than 80%, wherein organizing, 3 effects are best, and tumour cell returns
For yield up to 94%, group 1, group 5,6 tumor cell recoveries of group are low undesirable.
Although present invention has been a certain degree of description, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate change of each condition can be carried out.It is appreciated that the invention is not restricted to the embodiment, and it is attributed to right
It is required that scope, it includes the equivalent substitution of each factor.
Claims (10)
1. a kind of circulating tumor cell sorting and/or the method for enrichment, the described method comprises the following steps：
1) biotin labeling anti-Her2 (human epidermal growth factor receptor 2) monoclonal antibody is used, (epithelial cell glues anti-EpCAM
Attached molecule) monoclonal antibody and anti-Trop2 (human trophocyte cell's surface antigen 2) monoclonal antibody, and be mixed；
Wherein, the mixed mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 20-40% of biotin labeling；
The anti-EpCAM monoclonal antibody 10-25% of biotin labeling；
The anti-Trop2 monoclonal antibodies 40-60% of biotin labeling；
2) mixture for obtaining step 1) is added in cell liquid to be sorted；
Wherein, the volume ratio of the mixture and cell liquid is 1~5:1000；
3) magnetic bead of Streptavidin coupling is added, is incubated 20 minutes at 4 DEG C, magnetic bead is collected, produces；
Wherein, the volume ratio of the cell liquid to be sorted and magnetic bead is 1000:10~30.
2. according to the method for claim 1, it is characterised in that in step 1), after three kinds of monoclonal antibodies mixing
Mass percent scope be：
The anti-Her2 monoclonal antibodies 35% of biotin labeling；
The anti-EpCAM monoclonal antibody 20% of biotin labeling；
The anti-Trop2 monoclonal antibodies 45% of biotin labeling；
Preferably, in step 2), the volume ratio of the mixture and cell liquid is 2~3:1000；
Preferably, in step 3), the volume ratio of the cell liquid to be sorted and magnetic bead is 1000:20.
Preferably, the tumour is selected from breast cancer, cancer of the esophagus, non-small cell lung cancer, cervix cancer, colon cancer, stomach cancer, uterine cancer
And/or nasopharyngeal carcinoma.
3. method according to claim 1 or 2, it is characterised in that in step 1), the list of three kinds of biotin labelings
Clonal antibody is prepared by the method comprised the following steps respectively：
A. ultrafiltration post antibody purification solution is used；
B. NHS-PEG4-Biotin solution is added, reacts 0.5~4h at room temperature；
Wherein, the molar concentration ratio of NHS-PEG4-Biotin and antibody is 10-50:1；
0.5-3h is preferably reacted at room temperature；1h is more preferably reacted at room temperature；
C. the monoclonal antibody solution for remove free biotin, obtaining biotin labeling is isolated and purified；
Preferably, described isolate and purify is carried out using sephadex.
4. according to the method for claim 3, it is characterised in that the step a is completed by the method comprised the following steps：
1. prepare mark reaction solution；
The mark reaction solution includes following components：
NaCl：10-300mmol/L；KCl：1-5mmol/L；Na2HPO4：5-50mmol/L；KH2PO4：1-5mmol/L；
Preferably, the mark reaction solution includes following components：
NaCl：137mmol/L；KCl：2.7mmol/L；Na2HPO4：10mmol/L；KH2PO4：2mmol/L；
2. adding mark reaction solution in ultrafiltration post, monoclonal antibody is then added, is mixed, is then centrifuged for, abandons filtrate；
Wherein, the volume mass ratio of the label solution and the monoclonal antibody is 1:1-50(μl/μg)；
Preferably, repeat step 2~3 times；
3. mixing the liquid of the residual in ultrafiltration post, after being stored at room temperature, the reversion of ultrafiltration post is inverted in a new super filter tube, from
The heart collects filtrate, takes PBS to be mixed in ultrafiltration post, stands；Ultrafiltration post is inverted, filtrate is collected by centrifugation.
5. according to the method any one of claim 1-4, it is characterised in that in step 2), biotin labeling resists
Her2 monoclonal antibodies, the anti-EpCAM monoclonal antibody of biotin labeling, the anti-Trop2 monoclonal antibodies of biotin labeling
The final concentration that mixture uses in cell liquid is respectively 300 μ g/ml-600 μ g/ml；
Preferably, the anti-Her2 monoclonal antibodies of the biotin labeling, the anti-EpCAM monoclonal antibody of biotin labeling, life
The final concentration that the mixture of the anti-Trop2 monoclonal antibodies of thing element mark uses in cell liquid is respectively 500 μ g/ml.
6. a kind of kit for sorting and/or being enriched with for circulating tumor cell, the kit include the anti-of biotin labeling
The anti-Trop2 monoclonal antibodies of Her2 monoclonal antibodies, the anti-EpCAM monoclonal antibody of biotin labeling and biotin labeling
Mixture.
7. kit according to claim 6, it is characterised in that the mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 20-40% of biotin labeling；
The anti-EpCAM monoclonal antibody 10-25% of biotin labeling；
The anti-Trop2 monoclonal antibodies 40-60% of biotin labeling.
8. kit according to claim 7, it is characterised in that the mass percent scope of three kinds of monoclonal antibodies is：
The anti-Her2 monoclonal antibodies 35% of biotin labeling；
The anti-EpCAM monoclonal antibody 20% of biotin labeling；
The anti-Trop2 monoclonal antibodies 45% of biotin labeling.
9. the kit according to claim 7 or 8, it is characterised in that the kit also includes marked by streptavidin
Magnetic bead.
10. the method according to any one of claim 1-6 or the reagent according to any one of claim 7-9
Purposes of the box in sorting and/or enrichment cycles tumour cell.
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